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选用中国人群常见的MHCⅠ类分子HLA-A2.1和HLA-B51限制性恶性疟CTL抗原表位, 构建了单表位重组疫苗pcDNA3.1/tr和pcDNA3.1/sh, 再将上述表位DNA序列串联, 构建成双表位重组疫苗pcDNA3.1/ts. 将3种重组疫苗分别转染C1R/HLA-A2.1和K562/HLA-B51细胞后均证实表达. 单表位重组疫苗pcDNA3.1/tr和pcDNA3.1/sh的表达分别促进细胞表面HLA-A2.1和HLA-B51的稳定性. 免疫共沉淀实验证实上述表位促进细胞表面HLAⅠ类分子组装, 并提示特异性表位在细胞表面的递呈. 双表位重组疫苗pcDNA3.1/ts在以上两个不同细胞系中的表达与两个单表位微型基因的表达结果相似, 证明双表位肽的串联编码构型不影响肽链中不同表位的特异性抗原递呈. 这一研究结果为进一步设计具有广泛覆盖率的多表位恶性疟重组CTL疫苗提供了理论和实验基础. 与常规体外CTL实验不同, 本研究观察微表位基因在表达特定MHCⅠ类分子的人细胞中进行内源性抗原递呈的过程, 其方法更接近人体内环境, 对于人类CTL抗原表位的确定和人重组CTL表位疫苗免疫原性的预测具有重要意义.
The CTL antigen epitopes of HLA-A2.1 and HLA-B51 restricted falciparum malaria, which are common MHC class I molecules in Chinese population, were selected to construct single-epitope recombinant vaccine pcDNA3.1 / tr and pcDNA3.1 / sh, DNA sequence was used to construct recombinant eukaryotic expression vector pcDNA3.1 / ts.The recombinant plasmids were transfected into C1R / HLA-A2.1 and K562 / HLA-B51 cells respectively.The recombinant vaccine pcDNA3 .1 / tr and pcDNA3.1 / sh respectively promote the stability of HLA-A2.1 and HLA-B51 on the cell surface.Immunoprecipitation experiments confirmed that these epitopes promote the assembly of HLA class I molecules on the cell surface and prompted the specific table Position on the cell surface.The expression of the recombinant eukaryotic expression vector pcDNA3.1 / ts in the above two different cell lines was similar to that of the two single-epitope mini-genes, which proved that tandem epitope Type does not affect the specific antigen presentation of different epitopes in the peptide chain.The results of this study provide theoretical and experimental basis for the further design of CTL vaccine with extensive coverage of multiple epitopes of P. falciparum.Compared with the conventional in vitro CTL experiments, This study looked at individuals who express a specific MHC class I molecule as a microepitope gene The process of endogenous antigen presenting in cells is much closer to the environment of the human body and is of great significance for the determination of human CTL epitopes and the prediction of the immunogenicity of human recombinant CTL epitope vaccines.